Interferon-γ antagonists and therapeutic uses thereof

ABSTRACT

Polypeptides and multimeric polypeptides capable of binding interferon γ which are useful therapeutically in methods of treating interferon γ-related conditions or diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional 60/697,689 filed 8 Jul. 2005, which application is hereinspecifically incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention encompasses fusion proteins and multimers capable ofbinding and inhibiting human interferon gamma (IFNγ), as well astherapeutic uses of such polypeptides.

2. Description of Related Art

Interferon gamma (IFNγ) is a lymphokine produced by activatedT-lymphocytes. It is known as an immune stimulant because of its abilityto activate monocytes and macrophages towards cell killing in vitro (Leeet al. (1983) J Immunol 131:221-2823). U.S. Pat. No. 4,897,264 describesthree different types of human IFNγ receptors having molecular weights90-105 KDa, 140 KDa, and 95-115 KDa. EP 393 502 provides the full-lengthhuman IFNγRα sequence of 489 amino acids (SEQ ID NO:1-2) (also known asCD119). U.S. Pat. No. 5,221,789 describes a fragment of human IFNγreceptor alpha (IFNγRα) from position 54-70. Soluble IFNγRα proteins (EP393 502) and chimeric fusion proteins having a fragment of an IFNγRαreceptor fused to an immunoglobulin component (EP 614 981) have beendescribed. WO 95/16036 describes human IFNγ receptor beta (IFNγRβ)having 337 amino acids (SEQ ID NO:3-4) (also known as AF-1).

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention features a nucleic acid moleculeencoding an interferon γ-binding fusion protein (R1)_(x)−(R2)_(y)−F,wherein R1 comprises a fragment of the extracellular domain ofinterferon gamma receptor alpha (IFNγRα) component, optionally modified,R2 comprises the extracellular domain of interferon gamma receptor beta(IFNγRβ) component or a fragment or modified fragment thereof, F is afusion component, and x and y are each independently a positive integer≧1, for example between 1 and 10, preferably x and y are eachindependently 1. The wild-type human IFNγRα and IFNγRβ nucleic acid andamino acid sequences are shown in SEQ ID NO:1-2 and SEQ ID NO:3-4,respectively.

In one embodiment, R1 is amino acids 1-239 of SEQ ID NO:2 (includes asignal peptide at positions 1-17), optionally modified with one or moreamino acid substitutions, and R2 is amino acids 28-246 of SEQ ID NO:4,optionally modified with one or more amino acid substitutions.Optionally, (R1)_(x)−(R2)_(y)−F further comprises a signal sequence(SS).

The optional fusion component (F) comprises any component that enhancesthe functionality of the fusion polypeptide. Thus, for example, a fusioncomponent may enhance the biological activity of the fusion polypeptide,aid in its production and/or recovery, or enhance a pharmacologicalproperty or the pharmacokinetic profile of the fusion polypeptide by,for example, enhancing its serum half-life, tissue penetrability, lackof immunogenicity, or stability. In preferred embodiments, the fusioncomponent is selected from the group consisting of a multimerizingcomponent, a serum protein, or a molecule capable of binding a serumprotein.

When the fusion component is a multimerizing component, the fusioncomponent includes any natural or synthetic sequence capable ofinteracting with any other multimerizing component to form a higherorder structure, e.g., a dimer, a trimer, etc. In specific embodiments,the multimerizing component is selected from the group consisting of (i)an immunoglobulin-derived domain, (ii) an amino acid sequence between 1to about 500 amino acids in length, optionally comprising at least onecysteine residue, (iii) a leucine zipper, (iv) a helix loop motif, and(v) a coil-coil motif. In a specific embodiment, theimmunoglobulin-derived domain is selected from the group consisting ofthe Fc domain of IgG or the heavy chain of IgG. In another specificembodiment, the Fc domain of IgG is human FcΔ1 (a), an Fc moleculecomprising a mutation of the region involved in forming the disulfidebond with the light chain.

When the fusion component is a serum protein, the serum protein may beany serum protein or a fragment of a serum protein. When the fusioncomponent is a molecule capable of binding a serum protein, it may be asmall molecule, a nucleic acid, a peptide, or an oligosaccharide. Thefusion component may also be a protein such as FcγR1, ScFv, etc. Inpreferred embodiments, the fusion component is encoded by the nucleicacid that encodes the fusion polypeptide of the invention. In someembodiments, however, such as when the fusion component is anoligosaccharide, the fusion component is attached post-translationallyto the expressed fusion polypeptide.

The nucleic acid molecule of the invention may further optionallycomprise a signal sequence (SS) component. When a SS is part of thepolypeptide, any SS known to the art may be used, including synthetic ornatural sequences from any source, for example, from a secreted ormembrane bound protein. In a preferred embodiment, an ROR signalsequence is used (SEQ ID NO:5).

In specific embodiments, the invention features a nucleic acid moleculeencoding the fusion polypeptide hIFNγRα.hIFNγRβ.hFc (SEQ ID NO:7) orhIFNγRα.hIFNγRβ(C174S).hFc (SEQ ID NO:9). In a more specific embodiment,the nucleic acid molecule is SEQ ID NO:6 or SEQ ID NO:8.

In a related second aspect, the invention features a vector comprising anucleic acid molecule of the invention. In third and fourth aspects, theinvention encompasses expression vectors comprising the nucleic acidmolecules operatively linked to an expression control sequence, andhost-vector systems for the production of a fusion polypeptide thatcomprise the expression vector, in a suitable host cell; host-vectorsystems, wherein the suitable host cell is, without limitation, abacterial, yeast, insect, mammalian or plant cell, such as tobacco; oranimals such as cows, mice, or rabbits. Examples of suitable cellsinclude E. coli, B. subtilis, BHK, COS and CHO cells. Fusionpolypeptides modified by acetylation or pegylation are also encompassedby the invention.

In a fifth aspect, the invention features a method of producing a fusionpolypeptide of the invention, comprising culturing a host celltransfected with a vector comprising a nucleic acid molecule of theinvention, under conditions suitable for expression of the protein fromthe host cell, and recovering the polypeptide so produced.

In a sixth aspect, the invention features a fusion polypeptidecomprising from N- to C-terminus, (R1)_(x)-(R2)_(y)-F, wherein R1, R2,F, x and y are as described above. X and y are preferably each a numberbetween 1-10; preferably x and y are each 1. In specific embodiments,the fusion polypeptide is an amino acid sequence selected from the groupconsisting of SEQ ID NO:7 and 9. In particular embodiments, the cysteineresidue at position 174 of IFNγRβ may be mutated to another amino acid,such as for example, serine, to eliminate disulfide bond scrambling ofthe protein and improving the production quality of the protein. Inspecific embodiments, Cys174 is mutated to Ser, Ala, Val, or Met.

In a seventh aspect, the invention features a multimeric protein,comprising two or more fusion polypeptides of the invention. In aspecific embodiment, the multimeric protein is a dimer. The interferonγ-binding multimers of the invention are capable of binding interferon γwith an affinity of at least 10⁻⁸ M, preferably at least 10⁻⁹ M, evenmore preferably at least 10⁻¹⁰ M, as determined by assay methods knownin the art. Generally, the ability of the dimeric interferon γ-bindingprotein to inhibit (e.g., block) the biological activity of interferonγ, may be measured by bioassay. Bioassays may include luciferase-basedassays using an ISRE or GAS promoter element, interferon γ stimulationof cell lines such as HT29 to produce IP-10 and/or interferon γstimulation of peripheral blood lymphocytes to increase cell surfaceexpression of MHC class II molecules.

In an eighth aspect, the invention features pharmaceutical compositionscomprising a fusion polypeptide of the invention with a pharmaceuticallyacceptable carrier. Such pharmaceutical compositions may comprise amonomeric or multimeric polypeptide, or nucleic acids encoding thefusion polypeptide.

The interferon γ-binding multimers of the invention are therapeuticallyuseful for treating any disease or condition which is improved,ameliorated, or inhibited by removal, inhibition, or reduction ofinterferon γ. In a ninth aspect, the invention features a methodcomprising administering to a patient having or at risk of developing adisease or condition which is ameliorated, inhibited, or treated with aninhibitor of interferon γ (IFNγ) a therapeutically or preventativelyeffect amount of a pharmaceutical composition of the invention. In oneembodiment, the condition treated is inflammatory bowel disease, such asulcerative colitis or Crohn's disease. In another embodiment, thedisease or condition is insulin-dependent diabetes, systemic lupuserythematosus, thyroiditis, multiple sclerosis, fulminant hepatitis,allograft rejection, thrombosis and hemorrhage following generalizedShwartzman-type reaction, and Kawasaki disease (mucocutaneous lymph nodesyndrome), AIDS, rheumatoid arthritis, including juvenile rheumatoidarthritis, Addison's disease, diabetes (type I), epididymitis,glomerulonephritis, Graves' disease, Guillain-Barre syndrome,Hashimoto's disease, hemolytic anemia, lupus nephritis, myastheniagravis, pemphigus, psoriasis, psoriatic arthritis, atherosclerosis,erythropoietin resistance, graft versus host disease, transplantrejection, autoimmune hepatitis-induced hepatic injury, biliarycirrhosis, alcohol-induced liver injury including alcoholic cirrhosis,rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies including ankylosing spondylitis, or vasculitis.Although any mammal can be treated by the therapeutic methods of theinvention, the subject is preferably a human patient suffering from orat risk of suffering from a condition or disease which can be improved,ameliorated, inhibited or treated with a fusion polypeptide of theinvention.

In a further aspect, the invention further features diagnostic andprognostic methods, as well as kits for detecting, quantifying, and/ormonitoring interferon y with the fusion polypeptides of the invention.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to describe the methods and/ormaterials in connection with which the publications are cited.

Definitions

The term “affinity for” interferon y means that the multimeric fusionproteins of the invention binds the intended cytokine(s) with anaffinity of at least 10⁻⁸ M, preferably at least 10⁻⁹ M, even morepreferably at least 10⁻¹⁰ M as determined by assay methods known in theart, for example, surface plasmon resonance (BiaCore ™) analysis. Themultimeric fusion polypeptide of the invention is capable ofspecifically binding to interferon γ, with an affinity (Kd) of at leastabout 10⁻⁹ M, or preferably at least about 1.3×10⁻¹¹ M, as determinedsurface plasmon resonance.

The term “capable of specifically blocking interferon γ” means theinterferon γ-binding fusion polypeptides of the invention form multimersthat inhibit the biological activity of the target cytokines, asmeasured by bioassay. Bioassays may include luciferase-based assaysusing an ISRE or GAS promoter element, and/or interferon γ stimulationof cell lines such as HT29. “IC50” is defined as the concentration offusion protein required to inhibit 50% of the response to interferon γas measured in a bioassay. In a bioassay, such as the ISRE-luciferaseassay described below, the interferon γ-specific multimeric proteins ofthe invention exhibit an IC50 of at least 1×10⁻⁸ M, more preferably atleast 1×10⁻⁹ M, and most preferably with an IC50 of at least 1×10⁻¹⁰ M.

The terms “treatment”, “treating”, and the like are used herein togenerally include obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease, condition, or symptoms thereof, and/ormay be therapeutic in terms of a partial or complete cure for a diseaseor condition and/or adverse effect attributable to the disease orcondition. “Treatment” as used herein covers any treatment of a diseaseor condition of a mammal, particularly a human, and includes: (a)preventing the disease or condition from occurring in a subject whichmay be predisposed to the disease or condition but has not yet beendiagnosed as having it; (b) inhibiting the disease or condition, i.e.,arresting its development; or (c) relieving the disease or condition,i.e., causing regression of the disease or condition. The population ofsubjects treated by the method of the invention includes subjectssuffering from the undesirable condition or disease, as well as subjectsat risk for development of the condition or disease.

By the term “therapeutically effective dose” is meant a dose thatproduces the desired effect for which it is administered. The exact dosewill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, for example,Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, a “condition or disease” generally encompasses acondition of a mammalian host, particularly a human host, which isundesirable and/or injurious to the host. Thus, treating a condition ordisorder with an interferon γ-binding fusion polypeptide will encompassthe treatment of a mammal, in particular, a human, who has symptomsreflective of elevated or deleterious interferon γ, or who is expectedto have such decreased activation in response to a disease, condition ortreatment regimen. Treating an interferon γ-related condition or diseaseencompasses the treatment of a human subject wherein reducing interferony levels with the fusion polypeptide of the invention results inamelioration of an undesirable symptom resulting from the interferonγ-related condition or disease.

“Inflammatory bowel disease (IBD)” includes conditions such asulcerative colitis (UC) and Crohn's disease (CD). Although UC and CD andgenerally considered different diseases, they are both characterized bypatchy necrosis of the surface epithelium, focal accumulations ofleukocytes adjacent to glandular crypts, and an increased number ofintraepithelial lymphocytes (IEL), and thus may be treated as a singledisease group.

General Description

Interferon gamma (IFNγ), also known as immune interferon, is a member ofthe interferon family, which exhibits the antiviral andanti-proliferative properties characteristic of interferons-α, and -βbut, in contrast to those interferons, is pH 2 labile. Human IFNγincludes a family of related polypeptide molecules that comprise thefull-length human IFNγ (Gray et al. (1982) Nature 295:503-508), avariant lacking the first three N-terminal amino acids, and other aminoacid sequence variants.

IFNγ receptors have been purified from different human cells (Aguet etal. (1987) J. Exp. Med. 165:988-999; Novick et al. (1987) J. Biol. Chem.262:8483-8487; Calderon et al. (1988) Proc. Natl. Acad. Sci. USA85:4837-4841), including IFNγ receptor a (EP 614 981 A1) and IFNγreceptor β (WO 95/16036).

Nucleic Acid Constructs and Expression

The present invention provides for the construction of nucleic acidmolecules encoding interferon γ-binding polypeptides. As describedabove, the nucleic acid molecules of the invention encode modifiedfragments of the wild-type (or naturally occurring) human IFNγRα and/orIFNγRβ proteins. Accordingly, the nucleic acid molecules may be termed“recombinant”, “artificial”, or “synthetic” as they are not nucleic acidmolecules found in nature, e.g., not naturally occurring sequences, butare sequences constructed by recombinant DNA technology.

These nucleic acid molecules are inserted into a vector that is able toexpress the fusion polypeptides of the invention when introduced into anappropriate host cell. Appropriate host cells include, but are notlimited to, bacterial, yeast, insect, and mammalian cells. Any of themethods known to one skilled in the art for the insertion of DNAfragments into a vector may be used to construct expression vectorsencoding the fusion polypeptides of the invention under control oftranscriptional and/or translational control signals.

Expression of the nucleic acid molecules of the invention may beregulated by a second nucleic acid sequence so that the molecule isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression may be controlled by any promoter/enhancer elementknown in the art. Promoters which may be used to control expression ofthe chimeric polypeptide molecules may include any promoter known in theart.

Expression vectors capable of being replicated in a bacterial oreukaryotic host comprising the nucleic acid molecules of the inventionare used to transfect the host and thereby direct expression of suchnucleic acids to produce the fusion polypeptides of the invention.Transfected cells may transiently or, preferably, constitutively andpermanently express the polypeptides of the invention. When thepolypeptide so expressed comprises a fusion component such as amultimerizing component capable of associating with a multimerizingcomponent of a second polypeptide, the monomers thus expressedmultimerize due to the interactions between the multimerizing componentsto form a multimeric polypeptide (WO 00/18932, herein specificallyincorporated by reference).

R1 and R2 Components

Interferon γ binds to a common receptor composed of two subunits (IFNγRαand IFNγRβ). Naturally occurring wild-type IFNγRα protein is a 489-aminoacid protein having the amino acid sequence of SEQ ID NO:2. R1 is anIFNγRα-derived component having amino acids 1-239 SEQ ID NO:2, or amodified fragment thereof. Naturally occurring human wild-type IFNγRβprotein is a 337-amino acid protein having the amino acid sequence shownin SEQ ID NO:4. R2 is an IFNγRβ fragment having amino acids 28-246 ofSEQ ID NO:4, or a variant thereof. Optionally, either or both componentscan be further modified to provide fusion proteins with specificallydesired properties, such as, for example, improved solubility, reducedimmunogenicity, improved PK, improved production characteristics, and/orimproved ability to block interferon γ activity. One of skill in the artmay determine what types of modifications may be made to improve orconfer a desired characteristic, for example, see PCT/US05/006266,herein specifically incorporated by reference.

Fusion Components

The fusion proteins of the invention comprise a fusion component (F)that, in specific embodiments, is selected from the group consisting ofa multimerizing component, a serum protein, or a molecule capable ofbinding a serum protein. When F comprises a multimerizing component, itincludes any natural or synthetic sequence capable of interacting withanother multimerizing component to form a higher order structure, e.g.,a dimer, a trimer, etc. The multimerizing component may be selected fromthe group consisting of (i) a multimerizing component, (ii) a truncatedmultimerizing component, (iii) an amino acid sequence between 1 to about500 amino acids in length, (iv) a leucine zipper, (v) a helix loopmotif, and (vi) a coil-coil motif. When F is a multimerizing componentcomprising an amino acid sequence between 1 to about 500 amino acids inlength, the sequence may contain one or more cysteine residues capableof forming a disulfide bond with a corresponding cysteine residue onanother fusion polypeptide comprising an F with one or more cysteineresidues.

In a preferred embodiment, the multimerizing component comprises one ormore immunoglobulin-derived domains from human IgG, IgM or IgA. Inspecific embodiments, the immunoglobulin-derived domain is selected fromthe group consisting of the Fc domain of IgG or the heavy chain of IgG.The Fc domain of IgG may be selected from the isotypes IgG1, IgG2, IgG3,and IgG4, as well as any allotype within each isotype group. In aspecific embodiment, F is the Fc domain of IgG4 with Ser228 (Kabatnumbering) mutated to Pro to stabilize covalent dimer formation (Mol.Immunol. (1993) 30:105-108) and/or Leu235→Glu which eliminates residualeffector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933). In apreferred embodiment, F is the Fc domain of IgG1, or a derivativethereof which may be modified for specifically desired properties (see,for example, Armour et al. (2003) Mol. Immunol. 40:585-593; Shields etal. (2001) J. Biol. Chem. 276:6591-6604). In specific embodiments, thefusion polypeptide of the invention comprises one or two Fc domain(s) ofIgG1. In one embodiment, F is an Fc derived from IgG2 or IgG4.

In one embodiment, F is a serum protein or fragment thereof and isselected from the group consisting of α-1-microglobulin, AGP-1,orosomuciod, α-1-acid glycoprotein, vitamin D binding protein (DBP),hemopexin, human serum albumin (hSA), transferrin, ferritin, afamin,haptoglobin, α-fetoprotein thyroglobulin, α-2-HS-glycoprotein,β-2-glycoprotein, hyaluronan-binding protein, syntaxin, C1R, C1q achain, galectin3-Mac2 binding protein, fibrinogen, polymeric Ig receptor(PIGR), α-2-macroglobulin, urea transport protein, haptoglobin, IGFBPs,macrophage scavenger receptors, fibronectin, giantin, Fc,α-1-antichyromotrypsin, α-1-antitrypsin, antithrombin III,apolipoprotein A-l, apolipoprotein B, β-2-microglobulin, ceruloplasmin,complement component C3 or C4, Cl esterase inhibitor, C-reactiveprotein, cystatin C, and protein C. In a specific embodiment, F isselected from the group consisting of α-1-microglobulin, AGP-1,orosomuciod, α-1-acid glycoprotein, vitamin D binding protein (DBP),hemopexin, human serum albumin (hSA), afamin, and haptoglobin. Theinclusion of an F component may extend the serum half-life of theinterferon α/β-binding polypeptide of the invention when desired. See,for example, U.S. Pat. Nos. 6,423,512, 5,876,969, 6,593,295, and6,548,653, herein specifically incorporated by reference in theirentirety, for examples of serum albumin fusion proteins.

When F is a molecule capable of binding a serum protein, the moleculemay be a synthetic small molecule, a lipid or liposome, a nucleic acid,including a synthetic nucleic acid such as an aptomer, a peptide, or anoligosaccharide. The molecule may further be a protein, such as, forexample, FcγR1, FcγR2, FcγR3, polymeric Ig receptor (PIGR), ScFv, andother antibody fragments specific for a serum protein.

Optional Spacers

Components of the fusion proteins of the invention may be connecteddirectly to each other or be connected via spacers. Generally, the term“spacer” (or linker) means one or more molecules, e.g., nucleic acids oramino acids, or non-peptide moieties, such as polyethylene glycol, whichmay be inserted between one or more component domains. For example,spacer sequences may be used to provide a desirable site of interestbetween components for ease of manipulation. A spacer may also beprovided to enhance expression of the fusion protein from a host cell,to decrease steric hindrance such that the component may assume itsoptimal tertiary structure and/or interact appropriately with its targetmolecule. For spacers and methods of identifying desirable spacers, see,for example, George et al. (2003) Protein Engineering 15:871-879, hereinspecifically incorporated by reference. A spacer sequence may includeone or more amino acids naturally connected to a receptor component, ormay be an added sequence used to enhance expression of the fusionprotein, provide specifically desired sites of interest, allow componentdomains to form optimal tertiary structures and/or to enhance theinteraction of a component with its target molecule. In one embodiment,the spacer comprises one or more peptide sequences between one or morecomponents that are between 1-100 amino acids, preferably 1-25. In aspecific embodiment, the spacer is a two amino acid sequence, forexample SG, RS, TG, etc.

Inhibition of Interferon γ Biological Activity

The fusion proteins of the invention are capable of inhibiting thebiological activity of interferon γ with an IC50 (concentration offusion protein required to inhibit 50% of the response to interferon γ)of at least 1×10⁻⁸ M, more preferably 1×10⁻⁹ M and most preferably1×10⁻¹⁰ M in a luciferase assay. Other bioassays useful to determineIC50 are known to the art, including, for example, IFNγ stimulation ofperipheral blood lymphocytes and/or HT29 cells.

Therapeutic Uses

The fusion polypeptides of the invention are therapeutically useful fortreating any disease or condition which is improved, ameliorated,inhibited or prevented by removal, inhibition, or reduction ofinterferon γ. Interferon γ has been implicated in a variety of clinicalconditions, such as inflammatory bowel disease (IBD), such as ulcerativecolitis or Crohn's disease, insulin-dependent diabetes, systemic lupuserythematosus, thyroiditis, multiple sclerosis, fulminant hepatitis,allograft rejection, thrombosis and hemorrhage following generalizedShwartzman-type reaction, Kawasaki disease (mucocutaneous lymph nodesyndrome), AIDS, rheumatoid arthritis, including juvenile rheumatoidarthritis, Addison's disease, diabetes (type I), epididymitis,glomerulonephritis, Graves' disease, Guillain-Barre syndrome,Hashimoto's disease, hemolytic anemia, lupus nephritis, myastheniagravis, pemphigus, psoriasis, psoriatic arthritis, atherosclerosis,erythropoietin resistance, graft versus host disease, transplantrejection, autoimmune hepatitis-induced hepatic injury, biliarycirrhosis, alcohol-induced liver injury including alcoholic cirrhosis,rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies including ankylosing spondylitis, or vasculitis.Although any mammal can be treated by the therapeutic methods of theinvention, the subject is preferably a human patient suffering from orat risk of suffering from a condition or disease which can be improved,ameliorated, inhibited or treated with a fusion polypeptide of theinvention.

Combination Therapies

In numerous embodiments, the fusion polypeptides of the invention may beadministered in combination with one or more additional compounds ortherapies. For example, multiple fusion polypeptides can beco-administered, or one polypeptide can be administered in conjunctionwith one or more therapeutic compounds. A benefit of the combined use ofthe fusion polypeptide of the invention with a second therapeutic agentis that combined use can provide improved efficacy and/or reducedtoxicity of either therapeutic agent.

Methods of Administration

The invention provides methods of treatment comprising administering toa subject an effective amount of a fusion polypeptide of the invention.In a preferred aspect, the fusion polypeptide is substantially purified(e.g., substantially free from substances that limit its effect orproduce undesired side-effects). The subject is preferably a mammal, andmost preferably a human.

Various delivery systems are known and can be used to administer anagent of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intra-articular, intravenous, subcutaneous, intranasal,intraocular, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. Administration can be acute or chronic (e.g. daily,weekly, monthly, etc.) or in combination with other agents. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In another embodiment, the active agent can be delivered in a vesicle,in particular a liposome, in a controlled release system, or in a pump.In another embodiment where the active agent of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see, for example, U.S. Pat. No.4,980,286), by direct injection, or byuse of microparticle bombardment, or coating with lipids or cell-surfacereceptors or transfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination. Systemic expression may also be achieved by plasmidinjection (intradermally or intramuscularly) and electroporation intocells.

In a specific embodiment, the pharmaceutical compositions of theinvention are administered locally to an area in need of treatment; thismay be achieved, for example, and not by way of limitation, by localinfusion during surgery, topical application, e.g., by injection, bymeans of a catheter, or by means of an implant, the implant being of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, fibers, or commercial skin substitutes.

A composition useful in practicing the methods of the invention may be aliquid comprising an agent of the invention in solution, in suspension,or both. The term “solution/suspension” refers to a liquid compositionwhere a first portion of the active agent is present in solution and asecond portion of the active agent is present in particulate form, insuspension in a liquid matrix. A liquid composition also includes a gel.The liquid composition may be aqueous or in the form of an ointment.

In one embodiment, the pharmaceutical composition of the invention is asustained release composition. Sustained release formulations fordelivery of biologically active peptides are known to the art. Forexample, U.S. Pat. No. 6,740,634, herein specifically incorporated byreference in its entirety, describes a sustained-release formulationcontaining a hydroxynaphtoic acid salt of a biologically activesubstance and a biodegradable polymer. U.S. Pat. No. 6,699,500 hereinspecifically incorporated by reference in its entirety, discloses asustained-release formulation capable of releasing a physiologicallyactive substance over a period of at least 5 months.

Diagnostic and Screening Methods

The fusion polypeptides of the invention may be used diagnosticallyand/or in screening methods. For example, the fusion polypeptide may beused to monitor levels of interferon y during a clinical study toevaluate treatment efficacy. In another embodiment, the methods andcompositions of the present invention are used to screen individuals forentry into a clinical study to identify individuals having, for example,too high or too low a level of interferon γ. The fusion polypeptides ofthe invention can be used in methods known in the art relating to thelocalization and activity of interferon γ, e.g., imaging, measuringlevels thereof in appropriate physiological samples, in diagnosticmethods, etc.

The fusion polypeptides of the invention may be used in in vivo and invitro screening assays to quantify the amount of non-bound interferon γpresent, e.g., for example, in a screening method to identify testagents able to decrease the expression of interferon γ. More generally,the fusion polypeptides of the invention may be used in any assay orprocess in which quantification and/or isolation of interferon γ isdesired.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising a fusion polypeptide of the invention. Such compositionscomprise a therapeutically effective amount of one or more fusionpolypeptide(s), and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

The fusion polypeptide of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree amino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the fusion polypeptide that will be effective for itsintended therapeutic use can be determined by standard clinicaltechniques based on the present description. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. Generally, suitable dosage ranges for intravenous administrationare generally about 0.02-10 milligrams active compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 10 mg/kg body weight.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. The amount of compoundadministered will, of course, be dependent on the subject being treated,on the subject's weight, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician. Thetherapy may be repeated intermittently while symptoms are detectable oreven when they are not detectable.

Cellular Transfection and Gene Therapy

The present invention encompasses the use of nucleic acids encoding thefusion polypeptides of the invention for transfection of cells in vitroand in vivo. These nucleic acids can be inserted into any of a number ofwell-known vectors for transfection of target cells and organisms. Thenucleic acids are transfected into cells ex vivo and in vivo, throughthe interaction of the vector and the target cell facilitated by lipidmixes or electroporation. The compositions are administered (e.g., byinjection into a muscle) to a subject in an amount sufficient to elicita therapeutic response. An amount adequate to accomplish this is definedas “a therapeutically effective dose or amount.”

In another aspect, the invention provides a method of reducinginterferon γ levels in a human or other animal comprising transfecting acell with a nucleic acid encoding a polypeptide of the invention,wherein the nucleic acid comprises an inducible promoter operably linkedto the nucleic acid encoding the polypeptide. For gene therapyprocedures in the treatment or prevention of human disease, see forexample, Van Brunt (1998) Biotechnology 6:1149-1154.

EXAMPLES

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Construction of Interferon γ-Binding Fusion Polypeptides

To create the interferon γ-binding fusion polypeptideshIFNγRα.hIFNγRβ.hFc (SEQ ID NO:7) nucleic acid encoding amino acids1-239 (including signal sequence 1-17) of the human IFNγRα sequence (SEQID NO:2) and amino acids 28-246 of the human IFNγRβ sequence (SEQ IDNO:4) were ligated into an expression vector which contained the humanFc sequence, thus creating a fusion protein having the IFNγRα andIFNγRβ, and the hinge, CH2 and CH3 regions of human IgG1 from the N to Cterminus. Molecules created encoded hIFNγRα₍₁₋₂₃₉₎.hFc (SEQ ID NO:11),hIFNγRα₍₁₋₂₃₉₎. hIFNγRβ.hFc (SEQ ID NO:7),hIFNγRα₍₁₋₂₃₉₎.hIFNγRβ(C174S).hFc (SEQ ID NO:9),hIFNγRα₍₁₋₂₃₉₎.TG.hIFNγRβ.SG.hFc (SEQ ID NO:13),hIFNγRα₍₁₋₂₃₉₎.TG.hIFNγRβ(C174S).SG.hFc (SEQ ID NO:15). All sequenceswere verified by standard molecular biology techniques. The appropriatecoding sequence was subcloned into a eukaryotic expression vector usingstandard molecular biology techniques.

The interferon γ-binding fusion polypeptide variantshIFNγRα.hIFNγRβ(C174S).hFc (SEQ ID NO:9) andhIFNγRα₍₁₋₂₃₉₎.TG.hIFNγRβ(C174S).SG.hFc (SEQ ID NO:15) were created bysite-directed mutagenesis of the parent fusion polypeptide usingtechniques known to the art, and confirmed by sequencing.

Example 2 Determination of Interferon γ Binding Affinity

The affinity of the interferon γ-binding fusion polypeptides for humaninterferon γ was measured using a BIAcore 2000™ or BIAcore 3000™, asdescribed in WO 00/75319, herein specifically incorporated by referencein its entirety. Briefly, the IFNγ-specific polypeptides were producedas small-scale supernatants by transiently transfecting CHO cells, usingLipofectamine/LIPO Plus® (Life Technologies), with DNA constructsencoding the proteins. Supernatants were collected after 72 hours andprotein expression was measured by Western blotting with anti-human FcHRP-conjugated antibody (Promega) and visualized by ECL (Pierce).Briefly, 5.4×10⁵ CHOK1 cells per well of a 6 well tissue culture dishwere transfected using 1 μg of DNA and 5 μl of lipofectamine in OptiMEM™(Gibco). After 12 h the cells were washed with OptiMEM™ and 2 ml of CHOserum free medium (Gibco) was added. After 60 h and 72 h the media wascollected and centrifuged to remove cellular debris. Expression levelsfor the various interferon γ-binding fusion polypeptides are shown inTable 1. For the BiaCore™ analysis, the IFNγ-binding fusion polypeptidesfrom the transiently transfected CHO supernatants were captured onto thechip surface using anti-human Fc antibodies. Various concentrations ofhuman IFNγ are injected over the surface and the time course ofassociation and dissociation are monitored. Kinetic analyses using BIAevaluation software were performed to obtain the association anddissociation rate constants. The results obtained for the dimericconstructs are summarized in Table 1. The Kd of control IFNγRα(1-239)-Fcwas 6.0×10⁻⁹ M whereas the Kd for hIFNγRα.hIFNγRβ.hFc was 1.3×10⁻¹¹ M,an approximately 60-fold improvement in affinity. hIFNγRβ.SG.hFc showedno ability to bind IFNγ. Only R1-R2-Fc constructs (with or withoutC174S) behaved as traps, exhibiting an affinity improvement from theindividual component affinities. Unlike many receptor-based fusionprotein traps, other configurations such as R2-R1-Fc or R1-Fc-R2 werepoor inhibitors, having a Kd values greater than 10 nM. Molecules havingN-terminal deletions in R1 (pCTR 2890, 2891, 2892, 2847, 2982, 2900,2901, 2982) expressed poorly or exhibited reduced the affinity for IFNγ.The presence or absence of linkers did not affect the affinity of themolecules.

Example 3 Determination of IFNγ IC50 Values Using Luciferase Bioassays

IFNγ Inhibition assay. The HEK293/ISRE or GAS-luciferase bioassay wasused to determine the ability of the IFNγ-specific polypeptides of theinvention to block the activity of human IFNγ. Human embryonic kidney293 (HEK293) cells, were transiently transfected with an ISRE- orGAS-luciferase reporter plasmids. By placing an ISRE or GAS promoterelement upstream of the luciferase gene one can monitor IFNγ activity incells. For the assay, transiently transfected ISRE or GAS-luciferaseHEK293 cells were suspended at 1.25×10⁵ cells per ml in medium and 0.08ml of cells plated (10,000 cells per well) into the wells of a 96 welltissue culture plate. Plates were incubated for ˜16 hours at 37° C. in ahumidified 5% CO₂ incubator. IFNγ-specific polypeptides and recombinanthuman IFNγ at varying doses were separately mixed in a 96 well tissueculture dish. 0.026 ml of each of these mixtures were then added to the96 well plate (IFNγ-specific polypeptides added first) containing theISRE or GAS-luciferase cells such that the final concentration of IFNγis 4 pM and the final concentrations of the IFNγ-specific polypeptideranged from 0.017 pM to 30 nM. Control wells contain no IFNγ-specificpolypeptide. Plates were incubated at 37° C. for 6 hours in a humidified5% CO₂ incubator. After 6 hours, the plates were equilibrated to roomtemperature for ˜30 minutes and 130 μl of Steady-Glo® luciferasesubstrate (Promega) was added. Plates were incubated at room temperaturefor ˜10 minutes and then read on a Victor™ multilabel counter(Luminescence 1 sec/well). IC50s were measured which is a 50% reductionin IFNγ stimulated activity, then determined with a 4 parameter fitanalysis using Prism™ software (Graph Pad). Table 1 shows the bioassayIC50 values of the IFNγ polypeptides produced as CHO transientsupernatants, whose concentrations were determined by Western blotanalysis using PAGE under reducing conditions. IC50 values also showthat only the R1-R2-Fc construct (with or without C174S) shows anenhanced ability to block IFN. The IC50 values in the ISRE and GASluciferase assays for hIFNγRα.hIFNγRβ.hFc and hIFNγRα.hIFNγRβ(C174S).hFc are 1-5×10⁻¹⁰M, whereas the individual receptor constructs,hIFNγRα₍₁₋₂₃₉₎.SG.hFc or hIFNγRβ.SG.hFc, gave an IC50 value of 6-8×10⁻⁹M or undetectable blocking, respectively.

TABLE 1 Expression BiaCore ™ IC50 (M) PCTR Constructs ug/ml (M) ISRE GASIFNγRα.Fc constructs 2852 hIFNγRα₍₁₋₂₄₆₎.SG.hFc n/a 2827hIFNγRα₍₁₋₂₃₉₎.SG.hFc 15 6.00E−09 6.20E−09 7.90E−09 ConfigurationVariants 2842 hIFNγRα₍₁₋₂₃₉₎.TG.hIFNγRβ.SG.hFc 12 1.29E−11 0.72E−102.02E−10  1.0E−10  3.8E−10 2987 hIFNγRα₍₁₋₂₃₉₎.hIFNγRβ.hFc 2845hIFNγRα₍₁₋₂₃₉₎.TG.hIFNγRβ(C174S).SG.hFc 10 1.18E−10   4E−10 1.54E−104.52E−10 2988 hIFNγRα₍₁₋₂₃₉₎.hIFNγRβ(C174S).hFc 2843hIFNγRβ.TG.HifnγRα₍₁₋₂₃₉₎.SG.hFc 15 No binding 1.46E−08 9.80E−09 2844hIFNγRα₍₁₋₂₃₉₎.TG.hFc.ARA.hIFNγRβ 15 8.00E−07 1.60E−08 2843hIFNγRβ.TG.hFc.ARA.hIFNγRα₍₁₋₂₃₉₎ n/a hIFNγRβ.Fc trap constructs 2828hIFNγRβ.SG.hFc 15 No binding 2848 hIFNγRβ(C174S).SG.hFc 15 No BindinghIFNγRα.Fc deletion constructs 2890 hIFNγRα₍₂₃₋₂₃₉₎.SG.hFc 2 2.57E−094.60E−09 2891 hIFNγRα₍₂₄₋₂₃₉₎.SG.hFc 10 1.20E−08 2.10E−08 2892hIFNγRα₍₂₉₋₂₃₉₎.SG.hFc n/a 2847 hIFNγRα₍₃₃₋₂₃₉₎.SG.hFc 1 2982hIFNγRα_((1-239Δ173-180)).SG.hFc n/a 2900 hIFNγRα₍₄₂₋₂₃₉₎.SG.hFc n/a2901 hIFNγRα₍₅₂₋₂₃₉₎.SG.hFc n/a R1 deletion constructs in differentconfigurations 2959 hIFNγRα₍₂₃₋₂₃₉₎.TG.hIFNγRβ.SG.hFc 4 5.30E−111.90E−10 2960 hIFNγRα₍₂₄₋₂₃₉₎.TG.hIFNγRβ.SG.hFc 2 1.30E−09 3.20E−09 2851hIFNγRα₍₃₃₋₂₃₉₎.TG.hIFNγRβ.SG.hFc 2 2.40E−07 1.30E−08 2962hIFNγRα₍₂₃₋₂₃₉₎.TG.hFc.ARA.hIFNγRβ n/a 7.90E−10 1.00E−09 2961hIFNγRα₍₂₄₋₂₃₉₎.TG.hFc.ARA.hIFNγRβ 10 6.00E−09 1.10E−08 2951hIFNγRα₍₃₃₋₂₃₉₎.TG.hFc.ARA.hIFNγRβ <1 Mouse constructs 2983 mIFNγRα.mFcn/a 2984 mIFNγRβ.mFc n/a 3002 mIFNγRα.TG.mIFNγRβ.SG.mFc n/a

1. A fusion protein comprising the amino acid sequence of SEQ ID NO:7 or9.
 2. A multimeric protein comprising two of the fusion proteins ofclaim
 1. 3. A fusion protein comprising the amino acid sequence of SEQID NO:13 or
 15. 4. A multimeric protein comprising two of the fusionproteins of claim
 3. 5. A fusion protein comprising R1,R2 and F in theN-terminus direction, and optionally a spacer sequence between R1 andR2, or R2 and F, or both, wherein R1 comprising amino acids 1-239 of SEQID NO:2; R2 comprising amino acids 28-246 of SEQ ID NO:4, whereincysteine 174 of SEQ ID NO:4 is optionally changed to a different aminoacid; and F is a multimerizing component selected from the groupconsisting of the Fc domain if IgG and a heavy chain of IgG; and whereinsaid fusion protein has an IC50 of 1-5×10⁻¹⁰ M in inhibiting theactivity of human IFNγ as measured by a luciferase assay, or a Kd ofabout 1×10⁻¹¹ M for human IFNγ as measured by surface plasmon resonance,or both.
 6. A nucleic acid molecule encoding the fusion protein of claim5.
 7. A vector comprising the nucleic acid molecule of claim
 6. 8. Ahost-vector system comprising the vector of claim 7, in a suitable hostcall.
 9. A method of producing a fusion polypeptide, comprisingculturing the host-vector system of claim 8 under conditions suitablefor expression of the protein from the host call, and recovering thepolypeptide so produced.
 10. A multimeric protein comprising two of thefusion protein of claim 5.